Autism spectrum disorder (ASD) is a developmental disorder that emerges in the prenatal period, likely during the first weeks of brain development. Chromatin regulatory events in early brain development have been repeatedly implicated in ASD. Chromatin regulation in prenatal development differs in fundamental ways from chromatin regulation in adulthood, which has been an obstacle to understand ASD pathogenesis. Here, we will use telencephalic organoids derived from human iPSCs to assess the functional activity of regulatory elements we identified through the PsychENCODE project to begin to unravel chromatin and gene regulation during early stages of cortical development, including stages that are not commonly accessible using postmortem brain tissue. We will longitudinally map the activity of these elements at critical developmental transitions in both normal organoids and ASD organoids, fractioned in different cell types (progenitors and neurons), examine their functional disruption in ASD by assessing their enrichment in disease-associated variants and determine their target genes from chromatin conformation capture experiments. In Aim 1, we will use STARR- seq to map the activity of H3K27ac histone-associated putative enhancers in organoids mimicking early cortical development and will compare the STARR-seq enhancers with histone-based enhancers active in stem cells, prenatal and adult postmortem brain identified through PsychENCODE and Epigenome Road map projects. In Aim 2, we will use ATAC-seq and STARR-seq to identify and compare enhancer activity in organoids from ASD patients and controls across early development and in different cell types. For this, we will use a collection of iPSC lines we generated from families with ASD. In Aim 3, we will use capture Hi-C and RNA-seq to study the 3D chromatin organization and promoter-enhancer interactions and their effect on gene expression in ASD neural cells. We will then explore whether ASD-implicated enhancers harbor disease- associated mutations by intersection with Simons and MSSNG whole genome public databases sequence variants. Finally, in Aim 4, we will carry out detailed functional analyses on ASD-associated mutations found in the implicated enhancers. We will engineer mutations in control iPSC lines, compare pairs of isogeneic organoids with or without the mutations, and perform capture Hi-C to identify their target genes and RNA-seq to confirm their effect on gene expression. These studies will chart gene regulation in human prenatal forebrain, across stages and cell types, map enhancers that are differentially active in early neural development in autism and identify mutations that are putatively responsible for these alterations. The end results will be the identification of a network of interacting genes involved in the pathophysiology of ASD, and the genetic/epigenetic mechanism responsible for their altered function in the disorder.